Lymphocyte-Based Morphometric Test for Alzheimer&#39;s Disease

ABSTRACT

This invention provides methods for diagnosing Alzheimer&#39;s disease in a symptomatic human subject, and for determining whether a human subject is predisposed to becoming afflicted with Alzheimer&#39;s disease. These methods employ the steps of (a) culturing a subject&#39;s lymphocytes with a suitable basement membrane matrix to permit the lymphocytes to aggregate; (b) measuring the resulting lymphocyte aggregation; and (c) based on such measurement, either diagnosing Alzheimer&#39;s disease or determining a predisposition to it, as appropriate.

This application claims the benefit of U.S. Provisional Application No.62/479,630, filed Mar. 31, 2017, the contents of which are incorporatedherein by reference.

Throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

FIELD OF THE INVENTION

The present invention relates to lymphocyte aggregation-based methodsfor diagnosing Alzheimer's disease in a human subject, and fordetermining whether a human subject is predisposed to having Alzheimer'sdisease.

BACKGROUND OF THE INVENTION

Many data have been collected over the last 15 years indicating that thepathophysiology of Alzheimer's disease is not just related to the brain,but can also have systemic expression. For example, many of the criticalamyloid and tau enzymes can be found throughout the body. On this basis,accurate assays have been developed to test skin cells, for example, forAlzheimer's disease against cells from a variety of control patients.Some skin cell-based assays focus on targets such as PKCΕ and ERK_(1,2).

SUMMARY OF THE INVENTION

This invention provides a method for diagnosing Alzheimer's disease in asymptomatic human subject comprising the steps of (a) culturinglymphocytes from the subject with a suitable basement membrane matrixfor a duration and under conditions sufficient to permit the lymphocytesto aggregate; (b) measuring the resulting lymphocyte aggregation; and(c) comparing the measurement of step (b) with a suitable control,thereby determining whether the subject is afflicted with Alzheimer'sdisease.

This invention also provides a method for determining whether a humansubject is predisposed to becoming afflicted with Alzheimer's diseasecomprising the steps of (a) culturing lymphocytes from the subject witha suitable basement membrane matrix for a duration and under conditionssufficient to permit the lymphocytes to aggregate; (b) measuring theresulting lymphocyte aggregation;

and (c) comparing the measurement of step (b) with a suitable control,thereby determining whether the subject is predisposed to becomingafflicted with Alzheimer's disease.

This invention further provides a method for diagnosing Alzheimer'sdisease in a symptomatic human subject comprising the steps of (a)culturing lymphocytes from the subject with a suitable basement membranematrix for a duration and under conditions sufficient to permit thelymphocytes to aggregate; (b) measuring the resulting lymphocyteaggregation; and (c) determining whether the measurement of step (b)correlates with Alzheimer's disease in the subject.

Finally, this invention provides a method for determining whether ahuman subject is predisposed to becoming afflicted with Alzheimer'sdisease comprising the steps of (a) culturing lymphocytes from thesubject with a suitable basement membrane matrix for a duration andunder conditions sufficient to permit the lymphocytes to aggregate; (b)measuring the resulting lymphocyte aggregation; and (c) determiningwhether the measurement of step (b) correlates with the subject's beingpredisposed to becoming afflicted with Alzheimer's disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Increased B lymphocyte aggregation for the Alzheimer's disease case

(AD-Panels A, B) compared with the Age-matched Control case (AC-Panels Cand D) and a Non-Alzheimer's Demented case (Non-ADD-Panels E and F).

Examples of 4x images at 48 hours after plating on a thick layer ofMatrigel® (1.82 mm). Panels A, C and E are examples where the cellseeding density is 125 cells/μl, while Panels B, D and F are examplesfor a cells seeding density of 250 cells/μl.

FIGS. 2A and 2B

% Aggregate Area at 48 hours after plating in 4x images

(FIG. 2A) Doubling the seeding cell density from 125 cells/μl to 250cells/μl results in a larger increase in the % Aggregate Area for theAlzheimer's disease case (AD-blue squares) when compared withNon-Alzheimer's Demented (Non-ADD-green triangles) or Age-matchedControl (AC-purple circles). (FIG. 2B) The slopes and intercepts for thefit lines from A are represented here. The slope, representing theAggregation Rate, is approximately 10-fold higher in the AD case whencompared with the AC and Non-ADD cases.

FIG. 3

Aggregate ranking in 4x images for two cell seeding densities—125cells/μl and 250 cells/μl

Ranking curves in natural logarithmic format, i.e., Ln (Aggregate Area),for the AD case (Panel A) are above the ranking curves for the Non-ADD(Panel B) and

AD (panel C) for the two cell seeding densities. (Panel D) Elevatedranking curve for the AD case (purple) compared with the Non-ADD (green)and AC (blue) for the cell seeding density of 125 cells/μl (Panel E)Elevated ranking curve for AD case (purple) compared with the Non-ADD(green) and AC (blue) for the cell seeding density of 250 cells/μl(Panel F) The difference between the ranking curves for the two cellseeding densities, i.e., Ln(Aggregate Area)₂₅₀₋ Ln(Aggregate Area)₁₂₅,is elevated for the AD case (purple) in all rankings compared with theNon-ADD (green) and AC (blue).

FIG. 4

Increased B lymphocyte aggregation for the Alzheimer's disease case

(AD-Panels A and B) compared with the Age-matched Control case(AC-Panels C and D) and a Non-Alzheimer's Demented case (Non-ADD-PanelsE and F). Examples of 10x images at 48 hours after plating on a thicklayer of Matriger (1.82mm). Panels A, C and E are examples for the cellseeding density of 125 cells/μl while Panels B, D and F are examples forthe cell seeding density of 250 cells/μl.

FIGS. 5A and 5B

% Aggregate Area for 125 and 250 cells/μl at 48 hours after plating in10x images—better approximation

(FIG. 5A, top) Doubling the seeding cell density from 125 cells/μl to250 cellsμl results in a larger increase in the % Aggregate Area for theAlzheimer's Disease case (AD-blue squares) when compared withNon-Alzheimer's Demented (Non-ADD-green triangles) or Age-matchedControl (AC-purple circles). Y−axis=% Aggregate Area; x−axis=celldensity (cells/μl). (FIG. 5B, bottom) The slopes and intercepts for thefit lines from (FIG. 5A) are represented here. The slope, representingthe Aggregation Rate, is significantly higher in the AD case whencompared with the AC and Non-ADD cases. Y−axis=slope; x−axis=intercept.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In this application, certain terms are used which shall have themeanings set forth as follows.

As used herein, “diagnosing Alzheimer's disease”, with respect to asymptomatic human subject, means determining that there is greater than50% likelihood that the subject is afflicted with Alzheimer's disease.Preferably, “diagnosing Alzheimer's disease” means determining thatthere is greater than 60%, 70%, 80% or 90% likelihood that the subjectis afflicted with Alzheimer's disease. As used herein, the phrase“determining whether the subject is afflicted with Alzheimer's disease”is synonymous with the phrase “diagnosing Alzheimer's disease.”

As used herein, “determining whether a human subject is predisposed tobecoming afflicted with Alzheimer's disease” means determining thatthere is a greater than average likelihood that the subject will becomeafflicted with Alzheimer's disease during her or his lifetime. After theage of 65, one in five individuals will become afflicted withAlzheimer's disease, and after the age of 85, one in two individualswill become afflicted with Alzheimer's disease. Preferably, determiningthis predisposition means determining that there is at least a 20%likelihood that the subject will become afflicted with Alzheimer'sdisease during her or his lifetime.

As used herein, “Alzheimer's disease” means a concurrent affliction withthe following three symptoms: (i) dementia; (ii) amyloid plaques; and(iii) neurofibrillary tangles. Dementia can be diagnosed during life.Cerebral amyloid plaques and neurofibrillary tangles can, for example,be diagnosed during autopsy. This definition of Alzheimer's disease isthe one provided by the

National Institute of Neurological Disorders and Stroke (NINDS) of theNational Institutes of Health (NIH), and is known as the “goldstandard.”

As used herein, a human subject who is “symptomatic” for Alzheimer'sdisease is a subject displaying at least one symptom of the disease,i.e., one of dementia, amyloid plaques, and neurofibrillary tangles.Preferably, a human subject who is symptomatic for Alzheimer's diseaseis a subject displaying dementia. Conversely, a human subject who is“asymptomatic” for Alzheimer's disease is a subject who does not displayany symptom of the disease.

As used herein, the “human subject” can be of any age. In oneembodiment, the subject is 40 years old or younger. In anotherembodiment, the subject is 50 years old or younger. In a furtherembodiment, the subject is over 40 years old. In yet a furtherembodiment, the subject is over 50 years old, over 60 years old, over 70years old, over 80 years old, or over 90 years old.

As used herein, “lymphocytes” include, by way of example, B lymphocytes,T lymphocytes, and mixtures thereof. Preferably, the lymphocytes are Blymphocytes. Methods for obtaining lymphocytes from a subject's bloodare known, and include, for example, flow cytometry, Ficoll (ahydrophilic polysaccharide that separates layers of blood), and gradientcentrifugation. A flow cytometry-based method is exemplified below.Additionally, in the subject methods, the lymphocytes (e.g., Blymphocytes) can be used in immortalized or primary (i.e.,non-immortalized) form. Methods for immortalizing lymphocytes (e.g., Blymphocytes) are known, and include, for example, treating thelymphocytes with Epstein-Barr virus (“EBV”). An EBV-based method isexemplified below.

As used herein, a “suitable basement membrane matrix” is any solid orsemisolid matrix (e.g., a gel matrix) useful for permitting the growthof cells thereon and/or therein. Such cell growth includes, withoutlimitation, cell division, cell enlargement, change in cell morphology,and formation of cell-cell attachments. Such matrices are known andinclude, without limitation, Matrigel®, Collagen I and IV, laminin,heparan sulfate proteoglycans, and entactin. Matrigel® and othermatrices such as laminin and collagen facilitate cell growth.

As used herein, culturing lymphocytes with a suitable basement membranematrix “for a duration and under conditions sufficient to permit thelymphocytes to aggregate” is achieved, for example, by conducting theculturing (i) for a duration of at least 30 minutes; (ii) at atemperature and in a growth factor milieu permissive of cell growth; and(iii) at a cell density that, at the beginning of culturing, permits atleast some of the lymphocytes to be in physical contact with otherlymphocytes. In one embodiment, the culturing is conducted for at least30 minutes and no longer than 48 hours. In another embodiment, thetemperature and protein milieu permissive of cell growth is 37° C., RPMI1640 Medium with 10% fetal bovine serum (“FBS”) and 1% penicillin(“PS”), in the presence of growth factor-reduced (“GFR”) Matrigel®(Corning, New York).

As used herein, culturing lymphocytes “from” a subject means culturinglymphocytes originating from the subject, wherein prior to culturing,the lymphocytes either have or have not been manipulated (e.g.,isolated, immortalized and/or otherwise modified) following removal fromthe subject.

In a preferred embodiment, the subject methods employ contacting a knownquantity of matrix (e.g., 750 μl, 500 μl, 250 μl, 150 μl or 75 μl ofMatrigel® in an assay well) with a known quantity of solution thatcontains a known quantity of lymphocytes to be tested. Thislymphocyte-containing solution is described as having a particular“density” of lymphocytes per unit volume of solution (e.g., 125 cells/μlor 250 cells/μl). Once placed in contact with the matrix, lymphocytes inthis solution of known cell density can contact, and affix themselvesto, the matrix's surface and interior, to the extent the matrix'sporosity permits. Cell densities that, at the beginning of culturing,permit at least some of the lymphocytes to be in physical contact withother lymphocytes include, without limitation, densities between 50cells/μl and 500 cells/μl.

As used herein, lymphocyte “aggregation” means the physical attachmentof at least one lymphocyte to at least one other lymphocyte. Thisattachment can be covalent or non-covalent. Moreover, for example, theattachment can involve one or more cell-cell interactions such asmembrane adhesion, membrane fusion, and cell-cell fusion. It is notedthat under the conditions envisioned for the subject methods, lymphocyteaggregation occurs both with lymphocytes from a subject afflicted withAlzheimer's disease and with lymphocytes from an un-afflicted subject.Under these conditions, however, the degree of aggregation differsbetween such lymphocyte populations. This unexpected difference, atleast in part, underlies the utility of the subject methods.

As used herein, “measuring” lymphocyte aggregation can be performed, forexample, using any method by which combined two-dimensional aggregatearea can be determined, or by which combined aggregate volume can bedetermined. In a preferred embodiment, and as exemplified herein,lymphocyte aggregation is measured by determining the combinedtwo-dimensional aggregate area as a percent fraction of the total matrix area examined. For example, in this scenario, a lymphocyteaggregation measurement of “50% aggregate area” means that when thematrix-containing assay well is viewed vertically, aggregatedlymphocytes (that are readily visualized as such) occupy 50% of theassay well's area. This type of measurement does not factor in thecombined aggregate volume or the combined surface area of the aggregatesper se. In further embodiments, measuring lymphocyte aggregationincludes determining total aggregation at a single time point (e.g., at10 minutes, 20 minutes, 30 minutes, one hour, two hours, four hours,eight hours, 12 hours, 24 hours or 48 hours), and/or at multiple timepoints (e.g., at zero hours, 10 minutes, 20 minutes, 30 minutes, onehour, two hours, four hours, eight hours, 12 hours, 24 hours and 48hours). Thus, the measurement can be of total lymphocyte aggregation (asmeasured, for example, by aggregation area) at a given time point,and/or of the rate of lymphocyte aggregate formation as derived frommeasurements taken during at least two time points.

Also envisioned is measuring lymphocyte aggregation by, and/or inconjunction with, one or more of the following methods disclosed in U.S.Pat. No. 8,658,134 (incorporated herein by reference): (i) determiningan integrated score; (ii) determining the average aggregate area pernumber of aggregates; (iii) performing a cell migration analysis; (iv)performing a fractal analysis; and (v) performing a lacunarity analysis.In conjunction with one or more of these methods, the following stepsare also envisioned: measuring cell and/or aggregate morphologycharacteristics such as the presence or absence of big clumps, thepresence or absence of cells attached to the clumps, the presence orabsence of big clumps growing, the number of clumps, the presence orabsence of remnant edges from a previously formed network of the clumps,the number of cells migrating, the presence or absence of cells beingnear percolation, the number of cell clumps, the size of cell clumps,and the growth of cell clumps.

As used herein, a “suitable control” for performing the subject methodsrequiring same includes, without limitation, a positive control, anegative control, or one or more of each. For example, a positivecontrol could be lymphocyte aggregation data obtained by performing oneof the subject methods on lymphocytes obtained from a human subjectafflicted with Alzheimer's disease. A negative control could be, forexample, lymphocyte aggregation data obtained by performing one of thesubject methods on lymphocytes obtained from a human subject who is notafflicted with Alzheimer's disease (e.g., a subject without anycognitive symptoms, a subject afflicted with mild cognitive impairment,or a subject afflicted with non-Alzheimer's dementia). Importantly, itis envisioned that in some of the subject methods, control tests (e.g.,positive, negative and/or both) will be performed before, concurrentlywith or after testing the lymphocytes from the human subjects ofinterest. The data from such control tests can then serve as suitablecontrols. It is also envisioned that in other of the subject methods, nocontrol tests are performed before, concurrently with or after testingthe lymphocytes from the human subjects of interest. Instead, in eachsuch method, determining affliction with, or predisposition to,Alzheimer's disease (as applicable) can be achieved, for example, bycomparing the method's result with lymphocyte aggregation parameters(e.g., 50% area at a lymphocyte density of 250 cells/μl and a durationof 48 hours) having a known correlation with disease state orpredisposition, as applicable.

Embodiments of the Invention

This invention provides lymphocyte-based methods for diagnosingAlzheimer's disease in a human subject. It also provideslymphocyte-based methods for determining whether a human subject ispredisposed to becoming afflicted with Alzheimer's disease. The subjectmethods are based, at least in part, on the surprising discovery that asubject's lymphocytes can be used in a morphometric test to eitherdiagnose Alzheimer's disease or determine a predisposition toAlzheimer's disease, as applicable.

Specifically, this invention provides a first method, namely, a methodfor diagnosing Alzheimer's disease in a symptomatic human subjectcomprising the steps of (a) culturing lymphocytes from the subject witha suitable basement membrane matrix for a duration and under conditionssufficient to permit the lymphocytes to aggregate; (b) measuring theresulting lymphocyte aggregation; and (c) comparing the measurement ofstep (b) with a suitable control, thereby determining whether thesubject is afflicted with Alzheimer's disease.

In this first method and the second, third and fourth methods describedbelow, the lymphocytes can be any type of lymphocytes. Preferably, thelymphocytes are B lymphocytes. Also preferred are B lymphocytes that areimmortalized.

Basement membrane matrices suitable for use in this first method, andthe second, third and fourth methods described below, are numerous andcommercially available. In the preferred embodiment, the suitablebasement membrane matrix is Matrigel®.

In this first method and the second, third and fourth methods describedbelow, the lymphocyte density is high enough to permit the requiredaggregation, and low enough so that the aggregation does not exceed theamount permitting meaningful results. In one embodiment, the lymphocytedensity is between 50 cells/μl and 500 cells/μl . In another embodiment,the lymphocyte density is between 50 cells/μl and 100 cells/μl , 100cells/μl and 150 cells/μl , 150 cells/μl and 200 cells/μl , 200 cells/μland 250 cells/μl , 250 cells/μl and 300 cells/μl , 300 cells/μl and 350cells/μl , 350 cells/μl and 400 cells/μl , 400 cells/μl and 450 cells/μl, and 450 cells/μl and 500 cells/μl . In a further embodiment, thelymphocyte density is 50 cells/μl , 100 cells/μl , 150 cells/μl , 200cells/μl , 250 cells/μl , 300 cells/μl , 350 cells/μl , 400 cells/μl ,450 cells/μl or 500 cells/μl .

In this first method and the second, third and fourth methods describedbelow, the duration sufficient to permit the lymphocytes to aggregate islong enough to permit the required aggregation, and brief enough so thatthe aggregation does not exceed the amount permitting meaningfulresults. In the preferred embodiment, the duration sufficient to permitthe lymphocytes to aggregate is between 30 minutes and 48 hours. In afurther embodiment, the duration sufficient to permit the lymphocytes toaggregate is between 30 minutes and two hours, 30 minutes and fourhours, 30 minutes and eight hours, 30 minutes and 12 hours, 30 minutesand 24 hours, two hours and four hours, two hours and eight hours, twohours and 12 hours, two hours and 24 hours, two hours and 48 hours, fourhours and eight hours, four hours and 12 hours, four hours and 24 hours,four hours and 48 hours, eight hours and 12 hours, eight hours and 24hours, eight hours and 48 hours, 12 hours and 24 hours, 12 hours and 48hours, and 24 hours and 48 hours.

In a preferred embodiment of the first method, this invention provides amethod for diagnosing Alzheimer's disease in a symptomatic human subjectcomprising the steps of (a) culturing immortalized B lymphocytes fromthe subject with Matriger for between 30 minutes and 48 hours at alymphocyte density between 50 cells/μl and 500 cells/μl ; (b) measuringthe resulting lymphocyte aggregation; and (c) comparing the measurementof step (b) with a suitable control, thereby determining whether thesubject is afflicted with Alzheimer's disease.

This invention also provides a second method, namely, a method fordetermining whether a human subject is predisposed to becoming afflictedwith Alzheimer's disease comprising the steps of (a) culturinglymphocytes from the subject with a suitable basement membrane matrixfor a duration and under conditions sufficient to permit the lymphocytesto aggregate; (b) measuring the resulting lymphocyte aggregation; and(c) comparing the measurement of step (b) with a suitable control,thereby determining whether the subject is predisposed to becomingafflicted with Alzheimer's disease.

In this second method and the fourth method described below, the subjectmay be afflicted with a cognitive disability. In one embodiment, thisdisability is mild cognitive impairment. Alternatively, in this secondmethod and the fourth method described below, the subject is notcognitively impaired.

In a preferred embodiment of the second method, this invention providesa method for determining whether a human subject is predisposed tobecoming afflicted with Alzheimer's disease comprising the steps of (a)culturing immortalized B lymphocytes from the subject with Matriger forbetween 30 minutes and 48 hours at a lymphocyte density between 50cells/μl and 500 cells/μl ; (b) measuring the resulting lymphocyteaggregation; and (c) comparing the measurement of step (b) with asuitable control, thereby determining whether the subject is predisposedto becoming afflicted with Alzheimer's disease.

This invention further provides a third method, namely, a method fordiagnosing Alzheimer's disease in a symptomatic human subject comprisingthe steps of (a) culturing lymphocytes from the subject with a suitablebasement membrane matrix for a duration and under conditions sufficientto permit the lymphocytes to aggregate; (b) measuring the resultinglymphocyte aggregation; and (c) determining whether the measurement ofstep (b) correlates with Alzheimer's disease in the subject.

In this third method, measuring the resulting lymphocyte aggregationcomprises measuring the percent aggregate area, and determining whetherthat percent aggregate area correlates with the percent aggregate areaexpected in a subject afflicted with Alzheimer's disease.

In a preferred embodiment of the third method, this invention provides amethod for diagnosing Alzheimer's disease in a symptomatic human subjectcomprising the steps of (a) culturing immortalized B lymphocytes fromthe subject with Matriger for between 30 minutes and 48 hours at alymphocyte density between 50 cells/μl and 500 cells/μl ; (b) measuringthe resulting lymphocyte aggregation; and (c) determining whether themeasurement of step (b) correlates with

Alzheimer's disease in the subject.

Finally, this invention provides a fourth method, namely, a method fordetermining whether a human subject is predisposed to becoming afflictedwith Alzheimer's disease comprising the steps of (a) culturinglymphocytes from the subject with a suitable basement membrane matrixfor a duration and under conditions sufficient to permit the lymphocytesto aggregate; (b) measuring the resulting lymphocyte aggregation; and(c) determining whether the measurement of step (b) correlates with thesubject's being predisposed to becoming afflicted with Alzheimer'sdisease.

In this fourth method, measuring the resulting lymphocyte aggregationcan be achieved via any suitable approach. In the preferred embodiment,measuring the resulting lymphocyte aggregation comprises measuring thepercent aggregate area, and determining whether that percent aggregatearea correlates with the percent aggregate area expected in a subjectpredisposed to becoming afflicted with Alzheimer's disease.

In a preferred embodiment of the fourth method, this invention providesa method for determining whether a human subject is predisposed tobecoming afflicted with Alzheimer's disease comprising the steps of (a)culturing immortalized B lymphocytes from the subject with Matriger forbetween 30 minutes and 48 hours at a lymphocyte density between 50cells/μl and 500 cells/μl ; (b) measuring the resulting lymphocyteaggregation; and (c) determining whether the measurement of step (b)correlates with the subject's being predisposed to becoming afflictedwith Alzheimer's disease.

This invention further provides a kit for performing any of the subjectmethods, wherein the kit comprises, in separate compartments or a singlecompartment, (i) a growth medium (e.g., RPMI 1640 Medium with 10% FBSand 1% PS) and (ii) a basement membrane matrix (e.g., Matrige or other3D matrix such as collagen or laminin), wherein the growth medium andmatrix together permit lymphocyte aggregation under suitable conditions.

This invention still further provides a composition of matter comprisingaggregated lymphocytes (e.g., EBV-immortalized B lymphocytes) and asuitable basement membrane matrix (e.g., Matrigel®).

Also envisioned is the application of the instant invention to (i) anynon-lymphocyte peripheral blood mononuclear cell (PBMC) and (ii) anypluripotent stem cell (iPSC) derived from a reprogrammedEBV-immortalized B lymphocyte(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158714/; andhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596471/). In thisembodiment, such cells are treated, mutatis mutandis, as lymphocytes aretreated in this invention.

Further envisioned is the application of each of the instant diagnosticmethods, but without the step of explicitly comparing the subject'slymphocyte aggregation measurement with a suitable control. That is,this invention provides a version of each of the four instant diagnosticmethods wherein the subject's lymphocyte aggregation measurement eitherdoes or does not meet a pre-determined threshold indicative ofAlzheimer's disease.

This invention will be better understood by reference to the exampleswhich follow, but those skilled in the art will readily appreciate thatthe specific examples detailed are only illustrative of the invention asdescribed more fully in the claims which follow thereafter.

EXAMPLE 1 PBMC Preparation.

Whole blood, sampled in BD Vacutainer CPTTM Cell Preparation Tubes withsodium heparin (BD Bioscience, Franklin Lakes, NJ, USA), was diluted 1:2in PBS and gently layered over cold Ficoll-Paque PLUS (GE Healthcare,Little Chalfont, UK) and kept on ice before centrifugation for 20 min at900×g without brake at 18-20 °C., The cell layer on top of theFicoll-Paque PLUS consisting of peripheral blood mononuclear cells(PBMCs) was collected and diluted in PBS. PBMCs were centrifuged againfor 7 min at 450×g at 18-20 ° C. The supernatant was removed and cellswere re-suspended in 5 ml PBS and counted before they were centrifugedat 350×g for seven min at 18-20° C. Finally, cells were re-suspended infreezing medium (10% DMSO in heat-inactivated fetal bovine serum (FBS)(Nordic Biolabs AB, Taby, Sweden) and placed at −70° C. in a Mr.FrostyTM Freezing Container (Thermo Fisher) for at least three hoursbefore being transferred to liquid nitrogen for extended storage.

Flow Cytometry.

For each individual cell sample, 5×10⁶ PBMCs were quickly thawed at 37°C. and diluted in 40 ml cold wash buffer (PBS supplemented with 2.5% FBS(Life Technologies Ltd. Paisley, UK) and 0.1% sodium azide). The cellsuspension was centrifuged at 250×g for five min and after discardingthe supernatant, the pellet was re-suspended in 400 ml wash buffer. Eachsample was stained for one hour in a light-protected environment at 2°C. with titrated amounts of anti-CD19 labeled with Alexa Fluor 700 (BDBiosciences). Samples were analyzed using a BD LSR H Special OrderSystem, controlled by the BD FACSDiva 6.0 software (BD Biosciences). Apreliminary forward scatter (FSC) versus side scatter (SSC) gate wasused to identify lymphocytes and, depending on sample size, a total ofup to 100,000 in-gate events were recorded. All datasets were migratedto FlowJo 7.6.5 (Treestar Inc. Ashland, Oreg., USA) for further gatingand analysis.

Cells were cultured as per Coriell Cell Repository specifications, i.e.,at 37° C., in water jacket CO2 incubators in RPMI 1640 Medium, with 10%FBS and 1% PS. The Growth Factor Reduced (GFR) Matrigel® from Corningwas thawed overnight at +4° C. and 750 μl/well was used. A 30-minuteincubation at 37 ° C. was used before cell seeding.

EXAMPLE 2

The inaccuracy of existing diagnostic methods for Alzheimer's diseasehas made therapeutic intervention difficult. This is particularly trueregarding therapeutic intervention that is early enough to preventsignificant neuro-degeneration and cognitive dysfunction.

The instant invention provides accurate diagnostic methods for patientssuspected of having Alzheimer's disease. These methods involvequantitatively measuring aggregation rates of human immortalized Blymphocytes. A basis of this invention is elevated in vitro lymphocyteaggregation (i.e., the “biomarker” measured here) correlative withincreasing lymphocyte density in Alzheimer's disease.

This new biomarker was successfully validated with a different assaybased on the imbalances of a second, different biomarker, and showedcomplete diagnostic overlap with the second biomarker.

The lymphocytes tested using the subject methods were banked cells fromthe Coriell Cell Repository. The disease-related “pedigrees” of thesebanked cells were validated via autopsy and/or were geneticallyconfirmed as Alzheimer's patients (AD) or non-Alzheimer's dementedpatients (Non-ADD) and non-demented age-matched controls (AC).

Given the accuracy of the subject methods, they are valuable fordiagnosing Alzheimer's disease and thereby permitting timely therapeuticintervention.

1-34. (canceled)
 35. A method for diagnosing Alzheimer's disease in asymptomatic human subject comprising the steps of (a) culturing Blymphocytes from the subject with a suitable basement membrane matrixfor a duration and under conditions sufficient to permit the lymphocytesto aggregate; (b) measuring the percent aggregate area of the resultinglymphocyte aggregation; and (c) comparing the measurement of step (b)with a suitable control, thereby determining whether the subject isafflicted with Alzheimer's disease, wherein (i) the culturing step (a)comprises seeding the lymphocytes at a density between 125 cells/μ1 and250 cells/μl, and (ii) the duration sufficient to permit the lymphocytesto aggregate is selected from the group consisting of between 30 minutesand two hours, between 30 minutes and four hours, between 30 minutes andeight hours, between 30 minutes and 12 hours, between 30 minutes and 24hours, between two hours and four hours, between two hours and eighthours, between two hours and 12 hours, between two hours and 24 hours,between two hours and 48 hours, between four hours and eight hours,between four hours and 12 hours, between four hours and 24 hours,between four hours and 48 hours, between eight hours and 12 hours,between eight hours and 24 hours, between eight hours and 48 hours,between 12 hours and 24 hours, between 12 hours and 48 hours, andbetween 24 hours and 48 hours.
 36. The method of claim 35, wherein the Blymphocytes are immortalized.
 37. The method of claim 35, wherein thesuitable basement membrane matrix is Matrigel.
 38. The method of claim35, wherein the duration sufficient to permit the lymphocytes toaggregate is between 30 minutes and two hours.
 39. The method of claim35, wherein the duration sufficient to permit the lymphocytes toaggregate is between two hours and four hours.
 40. The method of claim35, wherein the duration sufficient to permit the lymphocytes toaggregate is between four hours and eight hours.
 41. The method of claim35, wherein the duration sufficient to permit the lymphocytes toaggregate is between eight hours and 12 hours.
 42. The method of claim35, wherein the duration sufficient to permit the lymphocytes toaggregate is between 12 hours and 24 hours.
 43. The method of claim 35,wherein the duration sufficient to permit the lymphocytes to aggregateis between 24 hours and 48 hours.
 44. A method for diagnosingAlzheimer's disease in a symptomatic human subject comprising the stepsof (a) culturing immortalized B lymphocytes from the subject withMatrigel for between 24 hours and 48 hours at a lymphocyte cell seedingdensity between 125 cells/μl and 250 cells/μl ; (b) measuring thepercent aggregate area of the resulting lymphocyte aggregation; and (c)comparing the measurement of step (b) with a suitable control, therebydetermining whether the subject is afflicted with Alzheimer's disease.45. The method of claim 35, wherein the lymphocyte cell seeding densityis 125 cells/μl.
 46. The method of claim 35, wherein the lymphocyte cellseeding density is 250 cells/μl.
 47. The method of claim 44, wherein thelymphocyte cell seeding density is 125 cells/μl.
 48. The method of claim44, wherein the lymphocyte cell seeding density is 250 cells/μl.